Development of a single vector system that enhances trans-splicing of SMN2 transcripts.
RNA modalities are developing as a powerful means to re-direct pathogenic pre-mRNA splicing events. Improving the efficiency of these molecules in vivo is critical as they move towards clinical applications. Spinal muscular atrophy (SMA) is caused by loss of SMN1. A nearly identical copy gene called SMN2 produces low levels of functional protein due to alternative splicing. We previously reported a trans-splicing RNA (tsRNA) that re-directed SMN2 splicing. Now we show that reducing the competition between endogenous splices sites enhanced the efficiency of trans-splicing. A single vector system was developed that expressed the SMN tsRNA and a splice-site blocking antisense (ASO-tsRNA). The ASO-tsRNA vector significantly elevated SMN levels in primary SMA patient fibroblasts, within the central nervous system of SMA mice and increased SMN-dependent in vitro snRNP assembly. These results demonstrate that the ASO-tsRNA strategy provides insight into the trans-splicing mechanism and a means of significantly enhancing trans-splicing activity in vivo.
Coady
Tristan H
TH
Department of Veterinary Pathobiology, Bond Life Sciences Center, University of Missouri, Columbia, Missouri, United States of America.
Baughan
Travis D
TD
Shababi
Monir
M
Passini
Marco A
MA
Lorson
Christian L
CL
D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D002490:Central Nervous System; D005347:Fibroblasts / Q000473:pathology; D006801:Humans; D051379:Mice; D023421:Models, Animal; D009134:Muscular Atrophy, Spinal / Q000235:genetics; D016372:RNA, Antisense / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics*; D055540:Survival of Motor Neuron 2 Protein / Q000235:genetics*; D020040:Trans-Splicing; D014162:Transfection
D016372:RNA, Antisense; D012333:RNA, Messenger; D055540:Survival of Motor Neuron 2 Protein